https://doi.org/10.52973/rcfcv-e34336
Received: 11/10/2023 Accepted: 05/11/2023 Published: 29/01/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34336
ABSTRACT
Mammary cancer is a disease that requires effective treatments.
Conventional chemotherapy, while effective, often causes harmful
side effects. In contrast, metronomic chemotherapy (mCHT), which
involves the continuous administration of low doses of anticancer
drugs, is presented as a less aggressive alternative. In this study, the
genotoxic impact of treatment with Cyclophosphamide and Meloxicam
under the mCHT approach was evaluated in ten canine (Canis lupus
familiaris) patients with mammary carcinoma after undergoing
mastectomy. The patients underwent monthly evaluations, including
chest X–rays, blood tests, and the alkaline comet assay to measure
genotoxic effects of the antineoplastic drugs. These results
were compared with those of a group that received conventional
chemotherapy. The results revealed that patients treated with mCHT
experienced signicantly lower levels of DNA damage compared to
those who received conventional chemotherapy. Furthermore, DNA
damage decreased over time during mCHT, suggesting that dogs
may have developed tolerance to the treatment. Blood parameters
remained stable in the mCHT–treated group, and X–rays showed no
signs of recurrence or metastasis. All dogs survived during the one–
year follow–up without mammary cancer recurrence. It is concluded
that mCHT with Cyclophosphamide appears to be a less aggressive
therapeutic option with a more favorable genotoxic prole in the
treatment of mammary cancer in dogs.
Key words: Mammary cancer; metronomic chemotherapy (mCHT);
cyclophosphamide; genotoxicity; mastectomy
RESUMEN
El cáncer de mama es una enfermedad que demanda tratamientos
efectivos. La quimioterapia convencional, aunque eficaz, con
frecuencia ocasiona efectos secundarios perjudiciales. En contraste,
la quimioterapia metronómica (mCHT), que implica la administración
continua de dosis bajas de fármacos anticancerígenos, se presenta
como una alternativa menos agresiva. En este estudio, se evaluó
el impacto genotóxico del tratamiento con ciclofosfamida y
meloxicam bajo el enfoque de mCHT en diez pacientes caninas (Canis
lupus familiaris) con carcinoma mamario después de someterse
a mastectomía. Las pacientes se sometieron a evaluaciones
mensuales, que incluyeron radiografías de tórax, análisis de sangre
y el ensayo cometa alcalino para medir efectos genotóxicos del
antineoplásico. Estos resultados se compararon con los de un grupo
que recibió quimioterapia convencional. Los resultados revelaron
que las pacientes sometidas a mCHT experimentaron niveles
signicativamente menores de daño al ADN en comparación con
las que recibieron quimioterapia convencional. Además, se observó
una disminución del daño al ADN con el tiempo durante la mCHT, lo
que sugiere que las perras podrían haber desarrollado tolerancia al
tratamiento. Los parámetros sanguíneos se mantuvieron estables
en el grupo tratado con mCHT, y las radiografías no mostraron
signos de recurrencia o metástasis. Todas las perras sobrevivieron
durante el año de seguimiento sin recurrencia del cáncer de mama.
Se concluye que la mCHT con ciclofosfamida parece ser una opción
terapéutica poco agresiva con un perl genotóxico más favorable
en el tratamiento del cáncer de mama en perras.
Palabras clave: Cáncer de mama; quimioterapia metronómica
(mCHT); ciclofosfamida; genotoxicidad; mastectomía
Assessment of metronomic chemotherapy–induced DNA damage in
peripheral blood leukocytes from canine mammary cancer patients using
the alkaline comet assay
Evaluación del daño en el ADN inducido por quimioterapia metronómica en leucocitos de sangre
periférica de pacientes caninos con cáncer de mama mediante el ensayo cometa alcalino
Lorena Elizabeth Chalco–Torres
1,2
* , José Atilio Aranguren–Méndez
2
, Ana Elizabeth Guerrero–López
1
, Mauro Nirchio–Tursellino
1
1
Universidad Técnica de Machala, Ecuador.
2
Universidad del Zulia, Facultad de Ciencias Veterinarias, Laboratorio de Genética Molecular, Venezuela.
*Autor para correspondencia: lchalco@utmachala.edu.ec
Canine mammary cancer: comet assay / Chalco-Torres et al. _______________________________________________________________________
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INTRODUCTION
Mammary cancer is the most common type of cancer in women
and the most common cause of cancer–related deaths Worldwide,
being currently one of the most commonly diagnosed cancers and
the fth leading cause of cancer–related deaths, with an estimated
2.3 million new cases Worldwide [1].
Mammary cancer cells grow uncontrollably and break away from
the primary tumor spreading through the bloodstream or lymphatic
system to other parts of the body, where they can form new tumors
or invade nearby tissues, a process known as metastasis [2, 3]. The
treatment plans for mammary cancer may vary depending on the
type and stage of the cancer, as well as the individual's overall health
and preferences. The most common treatment options for mammary
cancer include surgery, chemotherapy, radiation therapy, targeted
therapy, immunotherapy, stem cell or bone marrow transplant, and
hormone therapy [4].
Conventional chemotherapy is the use of cytotoxic drugs to
target rapidly proliferating cells, particularly when these cells, such
as cancer cells, are especially vulnerable due to abnormalities in
the mechanisms that control adaptive responses to stress and cell
death [5, 6, 7]. Metronomic chemotherapy (mCHT) is a new method
to administer low–dose of anticancer drugs on a continuous and/
or frequent regular schedule (such as daily or weekly), usually over
a long period of time, with the advantage of causing fewer side
effects than standard chemotherapy, rarely developing acquired
drug resistance, and being cost–effective due to the lower dose
used [8]. However, as it is novel, mCHT requires further research for
a better understanding of the mechanisms of action, in particular
with regard to the antiangiogenic effect, the immune response, the
direct effects on cancer cells, Deoxyribonucleic acid (DNA) damage
(genotoxic effects) and the identication of biomarkers that predict
the response of the organism and mechanisms of resistance to this
treatment modality [9].
Chemotherapeutics target rapidly dividing cancer cells by directly
or indirectly inducing DNA damage [10]. However, antitumor drugs
are indiscriminate and do not selectively damage the DNA of tumor
cells [11, 12, 13]. Conventional chemotherapy can damage DNA in
cancer cells resulting in mutations or genome instability, which is a
key feature of both cancer and aging [12, 14]. When DNA is damaged,
it may lead to (1) mutations, which promote cancer, or (2) reduced DNA
replication, DNA and/or Ribonucleic acid (RNA) synthesis, cell–cycle
halt, cellular senescence, or cell death, which hastens the aging
process and reduces the functional capacity of cells or organs [12, 15].
The single–cell gel electrophoresis (SCGE) or alkaline comet assay,
a rapid, visual and sensitive method for quantify and analyze DNA
damage at the individual cell [16, 17, 18], has been used to investigate
the DNA–damaging effects of anti–neoplastic drugs and radiation
used during cancer therapy [19]. The alkaline comet assay has been
successfully used to demonstrate substantial accumulation of
fragmented DNA due to chemotherapy and has also been employed
to assess pre– and post–treatment levels of in vivo DNA damage
in peripheral blood leukocytes of cancer patients undergoing
chemotherapy [20].
There are a variety of parameters for evaluating genetic damage
by comet assay. The length of the tail, the amount of DNA in the
tail, and the tail moment are the most often utilized parameters. As
tail length does not frequently alter after the tail is formed, it can
only be employed at low levels of DNA damage [21]. As the damage
is increased, the tail's intensity follows suit. The amount of DNA in
the comet tail, which has a linear relationship with the frequency
of breaking, is another relevant statistic but the most helpful and
commonly used parameter is the tail moment, which combines the
tail length and tail intensity into a single number [16].
The use of animal models in human pathology research serves as
a valuable tool for exploring disease mechanisms and testing the
therapeutic effects of new and future drugs [22, 23, 24, 25]. Canine
(Canis lupus familiaris) mammary tumors, bearing striking similarities to
human breast cancer in terms of primary tumors, metastases, clinical
presentation, and treatment approaches, make dogs an invaluable
model for human cancer research [26, 27, 28, 29, 30, 31, 32, 33, 34].
Notably, conducting trials with novel antitumor therapies in pet dogs
within a veterinary clinical setting enables the collection of serial
biologic samples and facilitates the investigation of dose, schedule, and
corresponding pharmacokinetic/pharmacodynamic relationships [35].
In this context, the current study aimed to assess DNA damage in
female Canis familiaris with mammary neoplasms undergoing mCHT
(Cyclophosphamide and Meloxicam) using a modied comet assay to
detect DNA single–strand breaks (SSB) and double–strand breaks (DSB).
MATERIALS AND METHODS
Study population
The study encompassed a diverse group of 10 female dogs, that
included both purebred and mixed–breed individuals. All subjects
were diagnosed with various types of mammary carcinoma, such
as papillary tubule, solid mammary, complex, and micropapillary.
The selection criteria included grade 1 and 2 tumors with staging
between 2–4, absence of metastasis history, suitability for surgery
(mastectomy), and eligibility for mCHT.
Treatment regimen
After mastectomy, mCHT was administered orally for a duration
of 3 months. Cyclophosphamide was used at a dose of 10 mg·m
2
every 48 hours (h). Furthermore, as part of the adjuvant treatment,
a dose of Meloxicam at 0.01 mg·kg
-1
was administered every 24 h for
a duration of three months.
Monitoring
The animals underwent monthly evaluations, which included
ventrodorsal and laterolateral projections chest radiographs (x–ray
univet 300 HF, SERIAL D.11.1513629.15.123. Multimage. Cavaria–Italy) for
the detection of thoracic metastases and a comet assay to assess the
genotoxic effects of chemotherapy. Furthermore, monthly analyses of
hematological and biochemical parameters (TABLE I) were conducted
both before and after chemotherapy to assess the general condition
of the patients, identify potential side effects, and evaluate treatment
response. The blood sample was obtained from the cephalic vein after
shaving and disinfecting the area. A 1 ml sample was collected in an EDTA
tube for hematology analysis, 2 ml in a heparin tube for the comet assay
test, and 5 ml without anticoagulant for blood chemistry assessment.
Comparison group
To enable meaningful comparisons, it was incorporated data from
an additional group of patients diagnosed with neoplasms. This
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group received conventional chemotherapy consisting of intravenous
Doxorubicin at a dose of 30 mg·m
2
over three cycles, each with 21 d
intervals. This inclusion in the study ensures a basis for thorough
comparative analysis.
Comet assay procedure
Blood samples were obtained from each patient using a syringe
containing 0.10 mg of heparin. Each sample consisted of 10 uL of
blood mixed with 160 uL of Low Melting Point Agarose at 37°C. The
comet assay procedure followed the protocol detailed by Lu et al.
[16]. After electrophoresis, plates were xed with 2 mL of absolute
methanol, stained with DAPI, and covered with coverslips.
Image capture
Comets were recorded using a 20X objective in an Olympus BX53
epiuorescence microscope (Olympus Corporation, Ishikawa, Japan),
equipped with an Olympus DP73 digital camera coupled to CellSens
Dimension Software (Olympus) for image acquisition. A minimum of
50 comets were analyzed for each individual.
Statistical analyses
Statistical analyses were performed following the recommendations
described in Sokal and Rohlf [36]. To compare the data of the
hematologic and biochemical parameters determined among periods
of treatment, a one–way analysis of variance was used. Blood values
that did not meet the assumptions of normality and homoscedasticity
were transformed: Hematocrit (HCT) was transformed by the
relation θ=arcsen p, where p is a percentage. For the discrete
variables (erythrocytes, leucocytes HB, HCM, MCV), the root square
transformation was used.
The image analysis was carried out using the ImageJ software
[37] along with the OpenComet plugins [38]. These tools allowed
for the determination of primary measurements, such as tail length
(TL) and the tail DNA percentage (Tail DNA %) which were used to
calculate the Tail Moment
TM
%Tail Length Tail DNA
100
=
#
^h
, as proposed by Tice
et al. [39]. In particular, the (Tail DNA %) measure is recommended
in the scientic literature, as it helps reduce variability in the results,
as indicated by Kumaravel et al. [40].
Since comet assay data did not meet the assumptions of normality
and homoscedasticity, the non–parametric statistical Mann–Whitney U
test was used for comparing two groups, and the Kruskal–Wallis test was
utilized for comparing multiple groups using Statgraphics Centurion.
RESULTS AND DISCUSSION
TABLE I presents the mean ± standard deviation for each parameter
related to basic biometry and blood chemistry in the patients,
covering their data from the study's initiation throughout the three–
month treatment period. The analysis of variance demonstrated no
signicant statistical differences (P>0.05) with respect to treatment
duration among the analytes.
Conventional chemotherapy, while effective in cancer treatment,
can induce various side effects due to its impact on both cancerous
and healthy cells. The adverse effects caused by intravenous
administration of Cyclophosphamide in conventional chemotherapy
include tachycardia, marked leukopenia, and hyponatremia [41], as
well as hemorrhagic cystitis [42, 43].
Cancer often leads to a deterioration in the patient's iron prole,
potentially causing anemia. This condition can adversely impact the
effectiveness of antineoplastic treatments and patient survival. To
address the development of anemia, interventions such as iron and
TABLE I
Mean ± standard deviation of hematological and blood chemistry parameters in patients before (0) and after (1, 2 and 3 months) treatment with mCHT
Treatment time (months)
Parameter
0 1 2 3 P F
Hematocrit (%) 42.90 ± 3.12 42.60 ± 3.80 41.60 ± 3.3 42.70 ± 3.06 0.8171 0.31
Total Solids (g·L
-1
) 86.40 ± 9.28 86.60 ± 6.75 89.10 ± 10.06 85.50 ± 7.47 0.8024 0.33
Hemoglobin (g·L
-1
) 127.00 ± 9.69 131.20 ± 12.25 121.20 ± 9.94 128.50 ± 7.76 0.1681 1.78
Erythrocytes (×10
12
·L
-1
) 6.46 ± 0.48 6.47 ± 0.56 6.30 ± 0.48 6.38 ± 0.48 0.8646 0.24
VCM (fL) 66.66 ± 0.08 66.63 ± 0.14 66.64 ± 0.13 66.67 ± 0.00 0.5165 0.00
HCM (pg) 19.65 ± 0.64 20.05 ± 0.70 19.29 ± 0.84 20.21 ± 1.38 0.1388 1.95
CHCM (g·L
-1
) 296.09 ± 11.93 305.33 ± 9.29 292.29 ± 14.99 303.20 ± 20.72 0.1896 1.68
RDW (%) 14.87 ± 0.81 14.88 ± 0.78 15.22 ± 1.02 15.10 ± 0.79 0.7554 0.40
Leukocytes (×10
9
·L
-1
) 11.60 ± 2.56 12.25 ± 5.59 11.12 ± 4.56 15.00 ± 5.93 0.3200 1.20
Platelets (×10
9
·L
-1
) 281.80 ± 69.62 299.60 ± 92.58 312.83 ± 95.56 343.90 ± 207.48 0.6357 0.57
Urea (mg·dL
-1
) 35.30 ± 9.47 40.40 ± 8.03 34.80 ± 12.95 32.30 ± 9.45 0.3347 1.17
Creatinine (mg·dL
-1
) 1.02 ± 0.18 0.92 ± 0.21 1.03 ± 0.19 0.89 ± 0.26 0.3921 1.03
Total proteins (g·dL
-1
) 7.93 ± 0.74 7.92 ± 0.55 8.02 ± 0.85 7.65 ± 0.74 0.7059 0.47
GOT/AST (U·L
-1
) 77.10 ± 75.36 51.00 ± 19.91 38.30 ± 19.33 55.20 ± 24.24 0.3445 1.14
GPT/ALT (U·L
-1
) 57.00 ± 41.12 61.10 ± 20.70 35.00 ± 19.71 170.30 ± 262.02 0.1094 2.16
Ca (mg·dL
-1
) 8.70 ± 0.75 8.72 ± 1.02 8.3 ± 0.60 8.08 ± 0.68 0.2110 1.58
FIGURE 1. Radiographic images were assessed in laterolateral projections of
the chest. Patient with three neoplastic mass (a) and rst(b), second (c), and (d)
thirds month of mCHT. (e) One year after completing mCHT treatment
b
c
d
e
a
Canine mammary cancer: comet assay / Chalco-Torres et al. _______________________________________________________________________
4 of 8
erythropoietin supplementation are employed [44]. Anemia can
result from tumor–related factors, including inammation, oxidative
stress, and systemic metabolic changes in cancer patients, and it
can be further exacerbated by the toxic effects of chemotherapy
[45]. Notably, studies using Cyclophosphamide and Doxorubicin in
conventional chemotherapy have reported a decrease in red blood cell
count before and during treatment, with a more pronounced reduction
during the second treatment cycle [46]. These ndings underscore
the importance of managing anemia to optimize both the ecacy of
antineoplastic treatments and overall patient outcomes. Nonetheless,
results here provided yielded no substantial disparities in any of the
scrutinized blood parameters (TABLE I). What's particularly striking
is that the metronomic regimen exhibited only a marginal effect on
these metrics, implying that mCHT utilizing Cyclophosphamide might
represent a milder treatment approach concerning its impact on blood
parameters. This underscores the potential benets of metronomic
chemotherapy in ameliorating specic treatment–related adverse
effects, ultimately contributing to improved patient comfort and
treatment tolerability.
Radiological monitoring of patients with a diagnosis of mammary
cancer before, during, and after metronomic chemotherapy treatment
is displayed in FIG. 1 (ventro–dorsal projections are not included).
Notably, the initial radiograph revealed well–dened neoplastic
masses, indicative of the primary tumor lesions (FIG. 1a). Following
tumor removal and three months of mCHT, subsequent radiographs
showed no evidence of new opacities or nodules, suggesting a
positive response to treatment in terms of controlling recurrence
or metastasis (FIGS 1b, 1c, and 1d). The decision to perform a one–year
follow–up radiograph (FIG. 1e) highlights the importance of long–term
monitoring. These ndings suggest that the treatment has been
effective in controlling the disease and contributing to the overall
well–being of the patient.
Referring to the Comet assay, TABLE II illustrate the results of a
comparative analysis of the assessed parameters before initiating
metronomic therapy treatment and after three months of treatment.
In all cases, the Kruskal–Wallis non–parametric test revealed highly
signicant differences (P<0.001) between the time points before and
after treatment. The results of the Mann–Whitney test, examining Tail
Length, Tail DNA %, and Tail Moment, are presented in TABLE III. In all
cases, the variations in values between metronomic and conventional
chemotherapy were found to be exceptionally statistically signicant
(P<0.0001).
MCHT and conventional chemotherapy represent distinct
approaches to cancer treatment. Conventional chemotherapy
employs higher drug doses at dened intervals, often based on bone
marrow recovery, with the goal of directly targeting rapidly dividing
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FIGURE 2. Medians along with their corresponding 95% condence intervals for
the parameters analyzed before and after three months of treatment in canine
patients undergoing mCHT
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tumor cells. In contrast, mCHT focuses on inhibiting angiogenesis
within tumor tissues by administering chemotherapeutic drugs at
continuous, lower doses, which generally result in fewer toxic side
effects and reduced dependency on supportive therapies [8, 47, 48].
Among the drugs used in mCHT, Cyclophosphamide plays a
signicant role. It belongs to the group of nitrogenous mustard
alkylating agents and primarily acts during the S phase of the cell
cycle, exerting its effects by interfering with transcription and
DNA replication processes [49, 50]. The DNA damage induced by
Cyclophosphamide has been observed in both in vitro and in vivo
studies, often assessed through the comet assay, a technique that
detects DNA strand breaks [51, 52, 53, 54, 55, 56, 57, 58].
Living organisms are continually exposed to sources of DNA
damage, both endogenous and exogenous, which can signicantly
impact health and contribute to various diseases [59]. Studies
consistently show that patients with mamary cancer exhibit notably
higher levels of initial endogenous DNA damage compared to healthy
individuals [59, 60, 61]. In response to these genetic challenges, cells
have evolved complex mechanisms to safeguard genome stability,
including base excision repair (BER), nucleotide excision repair (NER),
mismatch repair (MMR), homologous recombination (HR), and non–
homologous end–joining (NHEJ) [62, 63].
Based on the presented data, there is no apparent evidence of
a substantial genotoxic effect associated with the administered
treatment. Notably, despite a signicant increase in DNA damage
during the initial month of treatment, this damage markedly decreased
during the second month and eventually returned to initial endogenous
levels by the third month (FIG. 2). These ndings suggest the possibility
that patients developed increased tolerance to the low doses of
Cyclophosphamide administered as the treatment regimen progressed.
This enhanced tolerance may have facilitated the effective repair of DNA
damage resulting from the underlying disease or the treatment itself.
TABLE II
Comparison of Medians among treatment times (months)
for Tail length (TL), Tail DNA % (T–DNA%) and Tail Moment
(TM). Kruskal–Wallis Test Statistics (χ
2
), P–value
Parameter
Treatment time (months) Kruskal–Wallis test
0 1 2 3 χ
2
P–value
Tail Length 7.0 21.0 14.0 13.0 224.737
P<0.0001
Tail DNA % 8.02 12.1 11.49 8.23 137.705
P<0.0001
Tail Moment 0.976 2.553 2.301 1.035 137.666
P<0.0001
Sample Size 394 536 613 646
TABLE III
Comparing Tail Length, Tail DNA %, and Tail Moment
Between Metronomic and Conventional Treatments:
Post hoc Wilcoxon–Mann–Whitney Test, P–value
Parameter Metronomic Conventional W of Mann–Whitney P–value
Tail Length 13.0 30.0 356347,0
P<0.0001
Tail DNA % 9.14 18.12 368349,0
P<0.0001
Tail Moment 1.283 5.942 368383,0
P<0.0001
Sample Size 1954 235
Canine mammary cancer: comet assay / Chalco-Torres et al. _______________________________________________________________________
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The data presented in this study suggests that the population
of canine patients under investigation, diagnosed with mammary
cancer displayed evidence of effective DNA damage repair. Initially,
these patients exhibited low levels of endogenous DNA damage.
However, after one month of Cyclophosphamide treatment, there
was a notable increase in DNA damage levels, likely a consequence
of the treatment's mechanism of action.
The use of Cyclophosphamide in metronomic chemotherapy
(mCHT) appears to offer a less aggressive alternative concerning
its impact on blood parameters and genotoxic effects compared
to conventional chemotherapy. This potential presents signicant
advantages for patients in terms of their overall well–being and their
ability to tolerate the treatment. During the one–year clinical follow–
up, signicant outcomes were observed. Seven patients achieved a
complete response to treatment, while two showed a partial response.
Unfortunately, one patient was lost during this period, unrelated to
cancer, succumbing to hemolysis induced by canine Ehrlichiosis.
The combined treatment approach, including mastectomy and
mCHT, delivered notable benets for canine patients with mammary
carcinomas. Importantly, all patients survived and showed no
neoplastic recurrence within the one–year evaluation. To establish the
consistency and long–term sustainability of this trend among canine
mammary cancer patients undergoing mCHT, further investigation
with a larger sample size is needed to draw more comprehensive and
statistically signicant conclusions regarding treatment ecacy and
durability in the context of mammary carcinoma in canines.
CONCLUSIONS
The study highlights the potential advantages of utilizing
Cyclophosphamide in mCHT compared to conventional chemotherapy,
as evidenced by its milder impact on blood parameters and genotoxic
effects. Moreover, it suggests the effectiveness of DNA damage
repair in patients undergoing mCHT. The clinical outcomes are
encouraging, with seven patients achieving complete responses
and two demonstrating partial responses to the treatment. Notably,
all patients survived the one–year follow–up without any signs of
neoplastic recurrence. However, it is imperative to emphasize
the necessity for further research with a larger sample size to
establish the long–term consistency and sustainability of these
positive treatment results. This will allow for more comprehensive
and statistically signicant conclusions regarding the ecacy and
durability of mCHT in the context of canine mammary carcinoma.
ACKNOWLEDGEMENTS
This research was supported by Direction of Research, Development
and Innovation of Universidad Técnica de Machala (grant 2020/UTMACH‐
GPR‐GEN‐155 to M.N and grant PR–GEN–175 to L.E.C.T and A.G.E.L).
Conict of interests
The authors of this study declare that there is no conict of interest
with the publication of this manuscript.
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